The Skeletal Biology Section has continued to investigate the biochemical nature of stem cells that form hard tissues. Human post-natal bone marrow stromal stem cells (BMSSCs) have potent osteogenic capacity, offering great potential for cell transplantation therapy of bone defects. However, in contrast to hematopoietic stem cells or germ cells, BMSSCs have a more limited lifespan and progressively lose their ?stemness? during ex vivo expansion, possibly due to the lack of telomerase activity. We have forced the expression of human telomerase reverse transcriptase (hTERT) in BMSSCs by viral transduction, and found that this extended their lifespan and maintained their osteogenic potential. Interestingly, hTERT-expressing BMSSCs (BMSSCs-T) generated significantly more bone tissue, with a mineralized lamellar bone structure and associated marrow, when compared to control BMSSCs, in xenogeneic transplants. The enhanced bone forming ability of BMSSCs-T was correlated with a higher and sustained expression of the early pre-osteogenic stem cell marker, STRO-1, indicating that telomerase expression helped to maintain the osteogenic stem cell pool during ex vivo expansion. Our results are the first demonstration that telomerase expression can enhance the bone-forming capacity of human BMSSCs in vivo by overcoming critical technical barriers of ex vivo expansion and that telomerase therapy may provide a new strategy for bone regeneration or repair. We have also further characterized the self-renewal capability, multi-lineage differentiation capacity, and clonogenic efficiency of human dental pulp stem cells (DPSCs). DPSCs form ectopic dentin and associated pulp tissue in vivo. DPSCs were also found to be capable of differentiating into adipocytes and neural-like cells in vitro. Stromal-like cells were re-established in culture from primary DPSCs transplants and re-transplanted into immunocompromised mice to generate a dentin-pulp-like tissue, demonstrating their self-renewal capability. The odontogenic potential of twelve individual single-colony derived DPSC stains was determined. Two thirds of the single-colony derived DPSC strains generated abundant ectopic dentin in vivo, while only a limited amount of dentin was detected in the remaining one third. These results indicate that single-colony derived DPSC strains differ from each other with respect to their rate of odontogenesis. Taken together, these results demonstrate that DPSCs possess stem cell-like qualities including self-renewal capability and multi-lineage differentiation. To identify possible differences between progenitors of osteoblasts and odontoblasts at the level of transcription, we compared the gene expression profiles of bone marrow stromal stem cells (BMSSCs) and human dental pulp stem cells (DPSCs) as representative populations of osteoprogenitor cells and odontoprogenitor, respectively. Total RNA from primary cultures was reverse-transcribed to generate cDNA probes and then hybridized with the Research Genetics human gene Microarray filter GF211. The filter arrays were analyzed using the Research Genetics software package (Pathways). Human BMSSCs and DPSCs were found to have a similar level of gene expression for more than 4,000 known genes represented on the filter. A few differentially expressed genes including Insulin-like growth factor binding protein 7, and collagen type I alpha 2 were more highly expressed in BMSSCs while collagen type XVIII alpha 1, insulin-like growth factor 2, discordin domain tyrosine kinase 2, NAD(P)H menadione oxidoreductase, homolog 2 of Drosophila large disk and cyclin-dependent kinase 6 were highly expressed in DPSCs. Furthermore, we confirmed the differential expression of these genes by semi-quantitative PCR and Northern blot hybridization. The protein expression patterns for both IGF-2 and IGFBP-7, correlated with the differential mRNA levels seen between BMSSCs and DPSCs. Functional experiments also demonstrated that exogenous IGF-2 acts as a weak mitogen for BMSSCs, but does not affect differentiation. These results indicate that microarray filter technology can identify different patterns of gene expression between these two stem cell populations. This study provides a basis to further characterize the function roles of the differentially expressed genes in the development of dentin and bone. Recently, it has been determined that human adipose tissue presents an alternative source of multipotent stromal cells. We have defined the phenotype of the human adipose tissue-derived stromal cells in both the differentiated and undifferentiated states in comparison to human BMSSCs. Flow cytometry and immunohistochemistry show that human adipose tissue-derived stromal cells have a protein expression phenotype that is similar to that of human bone marrow stromal cells. Expressed proteins include CD9, CD10, CD13, CD29, CD34, CD44, CD 49, CD 49, CD54, CD55, CD59, CD105, CD106, CD146, and CD166. Expression of some of these proteins was further confirmed by PCR and immunoblot detection. Unlike human bone marrow-derived stromal cells, we did not detect the STRO-1 antigen on human adipose tissue-derived stromal cells. Cells cultured under adipogenic conditions uniquely expressed C/EBPalpha and PPARgamma, two transcriptional regulators of adipogenesis. Cells cultured under osteogenic conditions were more likely to be in the proliferative phases of the cell cycle based on flow cytometric analysis of PCNA and Ki67. The similarities between the phenotypes of human adipose tissue-derived and human bone marrow-derived stromal cells could have broad implications for human tissue engineering. Further studies are now aimed at determining the regulators that cause adipocytic conversion of human BMSSCs.